Project Summary/Abstract - CryoEM Guided Enhancement of Ribosome-Targeting Antibiotics The continued emergence of antibiotic resistance materially threatens modern medicine and by extension, modern society. A dearth of newly developed or discovered antibiotics has made the situation even more dire, as pan-resistant strains have emerged. Retooling and modifying existing classes of antibiotics offers an opportunity to study and counteract resistance mechanisms while producing truly rationally-designed small molecule drug leads. Iterative rounds of rapid structural characterization via cryoelectron microscopy (CryoEM) and synthetic modification offers a rational approach to such retooling efforts. One of the most prolific targets for antibiotics is the bacterial ribosome, which is inhibited by streptogramin antibiotics produced by several species of ?Streptomyces?. Streptogramin A (SA) binds at the peptidyl transferase center of the ribosome. Streptogramins are of limited value clinically because of resistance mechanisms including inactivation by acetyltransferases such as VatA and molecular interference by ribosomal protection proteins which displace the inhibitor. In collaboration with the Seiple laboratory at UCSF, we have access to a wide variety of streptogramin analogs. These are produced using modular synthesis, to allow rapid access to variations in structural and hydrogen bonding elements. Initially, we will use rounds of Minimum Inhibitory Concentration screening and structural characterization to increase the efficacy of streptogramin analogs for the ?E. coli ribosome. These will be followed by studies tuning the activity of streptogramin A analogs against bacteria expressing VatA, the acetyltransferase. Analogs with inhibitory effects will be characterized by CryoEM and acetylation rates will be measured to complement the resistance profiles. To probe the rise of acetylation-based resistance, we will then passage ?E. coli? expressing VatA in sub-MIC levels of SA analogs and sequence survivors. We will complement this by comprehensive mutation of VatA by deep mutational scanning, performing parallel competitive growth and deep sequencing. Together, these approaches will identify mutations that act as global stabilizers and mutations that provide substrate specificity, and guide efforts to enhance steric clashes between the SA analog and ?unmutable? residues identified. Following these studies, we will apply similar techniques to explore the structural basis of a ribosomal protection protein, EfrCD. Such proteins bear homology to efflux pumps, but lack the transmembrane domains required for cellular efflux. Explorations of the structural space within the ribosome in the presence of such protection proteins will help probe the poorly understood mechanisms of such resistance. Ultimately, the explosive growth of cryoEM coupled to modern modular synthesis provides an opportunity to retool antibiotic classes with limited clinical relevance by specifically modifying them to combat and understand mechanisms of antibiotic resistance.